lable at ScienceDirect

Regenerative Therapy 14 (2020) 215e221

Contents lists avai

Regenerative Therapy

journal homepage: http: / /www.elsevier .com/locate/reth

Original Article

Development of an efficient vitrification method for chondrocytesheets for clinical application

Asuka Hayashi a, Miki Maehara b, Ayuko Uchikura a, Hitomi Matsunari a, c,Kazuaki Matsumura d, Suong-Hyu Hyon e, Masato Sato b, c, Hiroshi Nagashima a, c, *

a Laboratory of Medical Bioengineering, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama, Kawasaki 214-8571, Japanb Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japanc Meiji University International Institute for Bio-Resource Research (MUIIBR), 1-1-1 Higashimita, Tama, Kawasaki 214-8571, Japand School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 Japane The Joint Graduate School of Veterinary Medicine, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-8580, Japan

a r t i c l e i n f o

Article history:Received 16 February 2020Received in revised form24 March 2020Accepted 14 April 2020

Keywords:ChondrocyteCell sheetVitrificationCryopreservationOsteoarthritis

Abbreviations: DMSO, dimethyl sulfoxide; EG, ethyserum; LN, liquid nitrogen; PBS, phosphate buffered* Corresponding author. Laboratory of Medical Bio

culture, Meiji University, 1-1-1 Higashimita, Tama, KaE-mail address: hnagas@meiji.ac.jp (H. NagashimaPeer review under responsibility of the Japane

Medicine.

https://doi.org/10.1016/j.reth.2020.04.0062352-3204/© 2020, The Japanese Society for Regener(http://creativecommons.org/licenses/by-nc-nd/4.0/).

a b s t r a c t

Introduction: Regenerative therapy using chondrocyte sheets is effective for osteoarthritis. The clinicalapplication of chondrocyte sheet therapy is expected to be further advanced by the use of a feasiblecryopreservation technique. Previously, we developed a chondrocyte sheet vitrification method; how-ever, it was too complex to be used for routine clinical application. Here, we aimed to develop a pro-totype method for vitrifying chondrocyte sheets for clinical practice.Methods: We developed a “circulating vitrification bag” as a container to process cell sheets for vitrifi-cation in an efficient and sanitary fashion. Moreover, we invented the “vitrification storage box”, which isuseful for the vitrification of cell sheets, long-term preservation, and transportation. These devices wereused to vitrify rabbit chondrocyte sheets, which were then assessed for their structural characteristicsand the viability of the component cells after rewarming.Results: In all cell sheet samples (n ¼ 7) vitrified by the circulating vitrification bag method, the integrityof the sheet structure was maintained, and the cell survival rate was similar to that of non-vitrifiedsamples (91.0 ± 2.9% vs. 90.0 ± 3.0%). Proteoglycan and type II collagen, which are major componentsof cartilage, were densely and evenly distributed throughout the chondrocyte sheet subjected to vitri-fication similarly to that observed in the non-vitrified sheet. After long-term storage using the vitrifi-cation storage box, the cell sheets maintained normal structure and cell viability (survival rate:81.2 ± 1.0% vs. 84.3 ± 1.8%) compared to the non-vitrified sheet.Conclusion: Our results indicate that the circulating vitrification bag method is an effective approach forrealizing the clinical application of vitrified chondrocyte sheets. The vitrification storage box is alsouseful for the long-term preservation of vitrified cell sheets, further enhancing the feasibility of theclinical application of cryopreserved chondrocyte sheets.© 2020, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This isan open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/

4.0/).

lene glycol; FBS, fetal bovinesaline.engineering, School of Agri-wasaki 214-8571, Japan.).se Society for Regenerative

ative Medicine. Production and ho

1. Introduction

Regenerative medicine using autologous chondrocyte sheetshas been reported to be effective for the treatment of osteoarthritis[1]. Allogeneic cell sheets created from polydactyly derived chon-drocytes obtained from polydactyly surgery were reported to havesimilar treatment effects as autologous chondrocyte sheets [2,3].

Cryopreservation of cell sheets would help expand the clinicalapplication of cell sheet therapy and promote its industrialization.We previously developed a vitrification method that allows for the

sting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221216

cryopreservation of chondrocyte sheets [4]. Through this method,cryopreserved chondrocyte sheets were found to maintain theircartilage repair ability [5]. However, this previous method involvescomplex work processes [4], and it would be desirable to improve itto develop a simpler and more practical method for clinical appli-cation. In addition, for the expansion of the use of chondrocytesheet therapy, it is important to establish technologies for the long-term preservation and transportation of cryopreserved cell sheets.To meet these requirements, we developed the “circulating vitri-fication bag” as a prototype device for allowing the practical use ofthe chondrocyte sheet vitrification method. Moreover, as a devicethat can maintain the stable vitrified state of cell sheets, the“vitrification storage box” was developed. The results of the per-formance of these devices based on the vitrification of rabbitchondrocyte sheets are reported.

2. Materials and methods

2.1. Preparation of rabbit chondrocyte sheets

Rabbit chondrocyte sheets were prepared according to ourprevious report [4]. Briefly, commercially available primarycultured cells derived from the knee cartilage of a Japanese whiterabbit (CHC04C; Cosmo Bio Co., Ltd., Hokkaido, Japan) were platedonto temperature-responsive culture dishes (UpCell®, diameter:35 mm; CellSeed, Tokyo, Japan) at a density of 5.9 � 104 to7.5 � 104 cells/dish and cultured in DMEM/F12 medium (11320;Thermo Fisher Scientific K.K., Tokyo, Japan) supplementedwith 20%fetal bovine serum (FBS; SH30070.03, GE Healthcare, Tokyo, Japan)at 37.5 �C in a humidified atmosphere of 5% CO2 in air. Uponreaching confluence, the medium in each dish was replaced withRPMI1640 medium (11875; Thermo Fisher Scientific K.K.) supple-mented with 10% FBS and 100 mM L-ascorbic acid (3140401A1039;FUJIFILM Pharma Co., Ltd., Tokyo, Japan). The cells formed a singlethin layer 2 weeks after plating, at which time the UpCell® disheswere placed at 25 �C for 30 min to promote detachment of the cellsheet from the bottom surface of the dish. The cell sheet was thenremoved from the dish using a cell shifter (CSD001; CellSeed). Twocell sheets were layered together to form a double-layered sheet,which was further cultured for 1 week.

2.2. Vitrification and rewarming solutions

Vitrification and rewarming solutions were prepared accordingto our previous report [4] (Table 1). HEPES (20mM)-buffered TissueCulture Medium-199 (05909; Nissui Pharmaceutical, Tokyo, Japan)supplemented with 20% calf serum (12133C; Sigma-Aldrich, St.Louis, MO, USA) was used as the basal solution. Dimethyl sulfoxide(DMSO; 13407-45, NACALAI TESQUE, Kyoto, Japan) and ethyleneglycol (EG; 15209-85, NACALAI TESQUE) were used as permeable

Table 1Composition of each solution and the time and temperature of each step.

DMSO (v/v%) EG (v/v%) COOH-

Solutions for pre-vitrification treatmentEquilibration solution-1 10 10 e

Equilibration solution-2 10 10 e

Vitrification solution-1 20 20 10Vitrification solution-2 20 20 10Solutions for post-vitrification treatmentRewarming solution e e e

Dilution solution e e e

Washing solution-1 e e e

Washing solution-2 e e e

COOH-PLL: Carboxylated poly-L-lysine; DMSO: Dimethyl sulfoxide; EG: Ethylene glycol;

cryoprotectants. Sucrose (30404-45; NACALAI TESQUE) andcarboxylated poly-L-lysine (Bio Verde, Kyoto, Japan) were used asnonpermeable cryoprotectants.

An equilibration solution consisting of 10% (v/v) DMSO and 10%(v/v) EG in basal solution and a vitrification solution containing 20%(v/v) DMSO, 20% (v/v) EG,10% (w/v) carboxylated poly-L-lysine, and0.5 M sucrose were prepared. A rewarming solution and a dilutionsolution containing 1 M and 0.5 M sucrose, respectively, wereprepared, and the basal solution was used as the washing solution.The vitrification solution was used at an ice-cold temperature (oncrushed ice). All other solutions were used at room temperature(24e27 �C).

2.3. Vitrification and rewarming procedures

2.3.1. Circulating vitrification bag methodAs a container for the vitrification of cell sheets, we developed

the circulating vitrification bag, which allows sequential flushing ofsolutions during the pre- and post-vitrification processes. As shownin Fig. 1a, the circulating vitrification bag is a sealable polyethylenebag to which inflow and outflow tubes for injecting or dischargingvarious solutions, respectively, are attached. The cell sheet wassandwiched between two nylon meshes (45 � 45 mm, 95 mmthread diameter, 140 mm opening diameter), and the four cornerswere fixed with a stapler. After inserting the cell sheet into thecirculating vitrification bag, the zipper was closed while removingair in the bag (Fig. 1c1). The circulating vitrification bag wasattached to the stage of an orbital shaker (05-450-213; SANSYO Co.,Ltd., Tokyo, Japan), and the solutions shown in Table 1were injectedsequentially using a 20-ml syringe (Fig. 1b) while shaking the bagwith the following conditions; rotation number: 15 rpm, rockingangle: 20�, turning angle: 360�, under room temperature(24e27 �C). First, 10ml equilibration solution-1 was injected via theinflow tube into the circulating vitrification bag (pre-equilibration;Fig. 1c2). The cell sheet was kept in equilibration solution-1 for5 min while shaking the bag (Fig. 1c3). Then, the solution wasdischarged from the outflow tube using a 20-ml syringe (Fig. 1c4).By using this same procedure, the cell sheet was exposed toequilibration solution-2 for 12.5 min. After discharging the solu-tion, vitrification solution-1 was injected while shaking the bag.After confirming the permeation of the solution into every part ofthe bag, the bag was removed from the shaker and placed on ice.After exposure of the cell sheet to vitrification solution-1 for 5 min(pretreatment with vitrification solution), the bag was attached tothe shaker once again, and the solution was discharged. Based onthe same procedure described above, vitrification solution-2 wasinjected into the bag, and the cell sheet was exposed to the solutionon ice for 7.5 min. After the discharge of the solution, the circulatingvitrification bag was held 1 cm above the surface of liquid nitrogen(LN) in a 1-L Styrofoam container and vitrified by exposure to the

PLL (w/v%) Sucrose (mol) Time (min) Temperature

e 5 RTe 12.5 RT0.5 5 IT0.5 7.5 IT

1 1 RT0.5 3 RTe 5 RTe 5 RT

IT: Ice-cold temperature; RT: Room temperature.

Fig. 1. The structure of the circulating vitrification bag and operation procedure. a: (Left) The structure of the circulating vitrification bag. An inflow tube and an outflow tube(polytetrafluoroethylene tube, inner diameter: 1.2 mm) are attached to the polyethylene bag (110 � 85 mm; film thickness: 0.063e0.064 mm). The inflow tube has 15 holes (holediameter: 0.6 mm) to allow quick inflow of the solutions (indicated by arrows). The outflow tube has 30 holes (hole diameter: 0.6 mm), allowing for smooth solution discharge(indicated by arrows). (Right) A photograph of the circulating vitrification bag. b: A circulating vitrification bag attached on an orbital shaker that was operated with the followingconditions; rotation number: 15 rpm, rocking angle: 20� , turning angle: 360� , under room temperature (24e27 �C). c: Operation procedure of the circulating vitrification bag. (1)Insert the cell sheet sandwiched between two nylon meshes into the circulating vitrification bag and close the opening. Then, attach the bag to the shaker. (2) While shaking thecirculating vitrification bag, inject the equilibration solution-1 via the inflow tube (arrows). (3) Keep the cell sheet in solution for a given amount of time (Table 1). (4) Discharge thesolution in the circulating vitrification bag from the outflow tube (arrows). Repeat steps (2) to (4) to successively inject and discharge the pre- and post-vitrification solutions shownin Table 1.

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221 217

vapor (approximately �150 �C). After exposure to the LN vapor forat least 1 min, the cell sheet was rewarmed. The circulating vitri-fication bag was placed on a heating plate at 45 �C. At the sametime, the bag was covered by a gel pack that had been preheated to38 �C for rapid rewarming. After 1 min, when the cell sheet hadcompletely rewarmed, the circulating vitrification bag wasattached again to the shaker. While shaking the circulating vitrifi-cation bag, the solutions for rewarming, dilution/removal of cryo-protectants, and washing (shown in Table 1) were successivelyinjected and discharged. The cell sheet was exposed to rewarmingsolution for 1 min, dilution solution for 3 min, washing solution-1for 5 min (prewash), and finally washing solution-2 for 5 min.Following the washing process, the cell sheet sandwiched betweennylon meshes was removed from the bag using forceps and placedinto a 100-mm dish. The cell sheet was then retrieved from thenylon meshes using forceps and immersed in 5 ml of Caþþ- andMgþþ- free phosphate buffered saline (PBS(�)) in a 60-mm dish.After checking for visible damage (cracks), the sheet was enzyme-treated for the cellular viability assessment.

2.3.2. Envelope methodFor comparison with the circulating vitrification bag method,

the envelope method that we reported previously [4] was used tovitrify and rewarm chondrocyte sheets. Briefly, a cell sheet was

successively immersed in 5 ml of the solutions used for pretreat-ment prior to vitrification (Table 1) in a 60-mm dish. The disheswith vitrification solution-1 and -2 were placed on ice. Whenpicking up the cell sheet using forceps, we treated the cell sheetwith extra care to avoid breaking it. After treating the cell sheetwith vitrification solution-2, it was placed on a 90 � 50 mm rect-angular piece of aluminum foil (thickness: 12 mm; MitsubishiAluminum Co., Ltd., Tokyo, Japan) using forceps. The aluminum foilwas folded to enclose the cell sheet [4] andwas held 1 cm above theLN surface in a 1-L Styrofoam container, and the cell sheet wasvitrified by exposure to vapor (approximately �150 �C). Afterexposure to LN vapor for more than 1 min, the cell sheet wasrewarmed.

For rapid rewarming, the cell sheet wrapped with aluminum foilwas placed on a heating plate at 38 �C, and it was covered by a gelpack that had been preheated to 38 �C. One minute later, thealuminum foil was carefully unfolded, and the cell sheet wasretrieved using forceps without breaking it and successivelyimmersed in 5 ml of the solutions used for rewarming, dilution/removal of cryoprotectants, and washing (Table 1) in a 60-mm dish.After washing, the cell sheet was immersed in 5 ml of PBS(�) in a60-mmdish, and after checking it for visible damage (cracks), it wasenzyme-treated for the cellular viability assessment.

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221218

2.4. Evaluation of the cells composing the cell sheet

The cell sheet viability assessment was conducted according tothe method used in our previous study [4]. Briefly, the cell sheetwas cut into 1e2 mm2 pieces with ophthalmic scissors and incu-bated in DMEM/F12 medium containing 2 mg/ml collagenase II(17101; Thermo Fisher Scientific K.K.) at 37 �C for 60 min to isolatethe component cells. The suspension of the isolated cells wascentrifuged at 1000 rpm for 5 min, and the precipitated cells wereresuspended in DMEM/F12 medium; the cell viability was deter-mined after trypan blue staining [viability (%) ¼ live cells/live anddead cells � 100].

2.5. Histological examination and immunohistochemical staining

Histological examination and immunohistochemical stainingwere performed according to our previous report [4]. Double-layered cell sheets were harvested after culture or cryopreserva-tion and fixed in 4% paraformaldehyde solution for 1 week. Thespecimens were embedded in paraffin, sectioned, and placed onglass slides. After deparaffinization and rehydration, the sectionswere stained for proteoglycan with 0.1% Safranin-O or immuno-stained with type II collagen antibody. For immunohistochemistry,the slides were incubated with a diluted primary anti-human typeII collagen antibody (F-57; Daiichi Fine Chemical, Toyama, Japan)for 16 h at 4 �C, followed by incubationwith the EnVisionþMouse/HRP secondary antibody (K4000; DAKO, Glostrup, Denmark) for1 h at room temperature. Finally, the sections were stained withdiaminobenzidine (K3466; DAKO) and counterstained with he-matoxylin. Coverslips were mounted onto slides and sealed withnail polish. The slides were then examined under amicroscope, andimages were captured (Biozero BZ-X710; KEYENCE, Osaka, Japan).

2.6. Experimental design

2.6.1. Verification of the circulating vitrification bag methodTo verify the performance of the newly developed circulating

vitrification bag method, a group of rabbit chondrocyte sheets werevitrified using this new method and the previously reported en-velope method. Fresh cell sheets were used as non-vitrified con-trols. The sheet structure, viability of cells composing the sheet, anddistributions of proteoglycan and type II collagen, which are majorcomponents of cartilage, were examined in vitrified and non-vitrified control sheets.

2.6.2. Long-term preservation of cell sheets using the vitrificationstorage box

For the long-term storage and handling of vitrified cell sheets,which are vulnerable to physical damage due to devitrification, wedeveloped a vitrification storage box (Fig. 2aec). As shown inFig. 2b, the box is a thin stainless steel container with built-in LNabsorbents in the main body and the lid. In this experiment, weaimed to demonstrate that the vitrification storage box allows forlong-term storage of the rabbit chondrocyte sheet. The cell sheetswrapped with aluminum foil following pretreatment with cryo-protectants were vitrified in accordance with the envelope method[4] using the vitrification storage box. The vitrification storage boxcontaining the vitrified sheet was transferred to and stored in LNvapor (�150 �C) in an LN container (Fig. 2d) for a storage period ofone or six months. The cell sheets after the long-term storage pe-riods were compared with the cell sheets rewarmed immediatelyafter vitrification (vitrified controls) and fresh cell sheets (non-vitrified controls) in terms of the cell sheet structure and cellularviability.

3. Results

3.1. Characteristics of post rewarming rabbit chondrocyte sheetsvitrified with the circulating vitrification bag method

The circulating vitrification bag method was used to vitrifyrabbit chondrocyte sheets, and the integrity of the sheet structureas well as the viability of the component cells were examined afterrewarming. All of the vitrified sheets (n ¼ 7) showed no visibledamage and had the same appearance as non-vitrified cell sheets(Fig. 3aec), and the average cell viability was 91.0 ± 2.9% (Table 2).This cell survival rate was equal to that of the cell sheets vitrified bythe envelope method (n ¼ 7; 89.5 ± 1.4%) and the non-vitrifiedcontrol (n ¼ 7; 90.0 ± 3.0%).

In addition, portions of the cell sheets recovered after vitrifica-tion and rewarmingwere isolated to investigate the distributions ofproteoglycan and type II collagen using histochemical staining andimmunostaining. Similar to the cell sheet vitrified with the enve-lope method and the non-vitrified cell sheet, the cell sheets vitri-fied with the circulating vitrification bag method showed strongsafranin-o staining throughout the tissue section (Fig. 3def).These results indicated that proteoglycan was densely and evenlydistributed throughout the chondrocyte sheet. Moreover, the cellsheet also showed an even and abundant distribution of type IIcollagen (Fig. 3gei). These data showed that the extracellular ma-trix of the cell sheets had been maintained after cryopreservationwith the circulating vitrification bag method.

3.2. Post rewarming structure and cellular viability of the cell sheetstored for long periods using the vitrification storage box

The vitrification storage box was used to vitrify and store rabbitchondrocyte sheets for long periods, and the sheet structure andcellular viability of post rewarming cell sheets were assessed(Table 3). There was no visible damage in the vitrified cell sheetsstored for both 1 and 6 months in LN vapor in the LN container(Fig. 4). Furthermore, the rates of cell survival after storage for oneand six months (79.2 ± 2.6% and 81.2 ± 1.0%, respectively) wereequal to those of the cell sheets rewarmed immediately aftervitrification (80.1 ± 2.5%) and the non-vitrified controls(84.3 ± 1.8%).

4. Discussion

Vitrification techniques permit cryopreservation of cells andtissues with a high concentration of cryoprotectants. The benefit ofvitrification techniques is that the solution turns into a glassy(amorphous) state at ultralow temperatures without the formationof ice crystals that cause physical damage to the cells [6]. In thevitrificationmethod, samples are exposed to high concentrations ofcryoprotectants. During the pretreatment process of cell sheetvitrification, samples are exposed to various solutions to allowcryoprotectants to permeate into the cells in a stepwise fashion atincreasing concentrations between the isotonic solution and thevitrification solution, which has an osmolarity of 11000 mOsmol.Next, in the post-vitrification process, the cell sheet in ultra-hypertonic vitrification solution is then exposed in a stepwisefashion to solutions with decreasing concentrations, during whichcryoprotectants are diluted and eliminated. To complete theseprocesses, the previously reported vitrificationmethod [4] requireda cumbersome, manual process to transfer the cell sheet succes-sively from one solution to another; this method is unrealistic toimplement in actual clinical settings.

In contrast, the circulating vitrification bagmethod developed inthe current study allows the cell sheet to be exposed successively to

Fig. 2. The structure and operation procedure of the vitrification storage box. The vitrification storage box (a) and its internal structure (b). b: The main body of the vitrificationstorage box (75 � 75 � 12 mm) and its lid (75 � 75 � 5 mm) are both made of stainless steel perforated plates (1 mm in thickness), and they are connected by a hinge. Both the mainbody and the lid have a built-in liquid nitrogen (LN) absorbent. c: Three vitrification storage boxes can be accommodated in a stainless storage rack (76 � 78 � 57 mm). The storagerack can be accommodated in an LN container canister (d-3). d: The operation procedure of the vitrification storage box. (1) Place the cell sheet wrapped in the vitrification packageon the perforated plate of the main body of the box that was immersed and cooled in LN. (2) Vitrify the cell sheet in the vitrification storage box by exposing it to LN vapor (�150 �C)discharged from the LN absorbents. (3, 4) Place up to three vitrification storage boxes in a canister using the storage rack (3) and store them in LN vapor in the LN container (4). Thevitrification storage box was prepared on a special order by Umihira Co. Ltd., Kyoto, Japan.

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221 219

various solutions with different components and osmolarity bykeeping the cell sheet in a sealed bag while successively replacingthe solution in the bag. The circulating vitrification bag methodminimizes the number of occasions the operator comes in contactwith the cell sheet, which is helpful in reducing labor andenhancing sterility when applying the cell sheet vitrificationtechnique in clinical settings.

The main concern in the application of this new method wasthat the residual solution in the preceding step within the bag maynot be completely replaced by the solution in the following step.However, in our preliminary experiment, the solution in the bagwas confirmed to be almost completely replaced by the new so-lution after just one round of preflushing (Suppl. Fig. 1). It can beinterpreted that, in the circulating vitrification bag method, the cellsheet was properly exposed to each solution in the vitrifying-rewarming process, which allowed it to achieve similar perfor-mances as a sheet treated by the conventional method that requiresmanual handling of cell sheets.

In the cell sheet vitrification procedure, cells are exposed tolarge changes in the osmolarity of the solutions used. Rapidchanges in osmolarity can be a stress factor for cells [7]. However,

the use of the circulating vitrification bag is thought to be beneficialfor reducing the osmotic stress to cells during cell sheet treatment.The reason for this is that in the circulating vitrification bagmethod, the cell treatment solution used in the preceding step isgradually replaced by the solution in the subsequent step(Suppl. Fig. 1). Therefore, the change in osmolarity during thisprocess is estimated to be milder. In other words, a well-designedsolution replacement method can minimize the stress due to os-motic changes while improving the survival rates of the componentcells of vitrified cell sheets. As the bag used in this study was aprototype, the optimization of both the inflow and outflow rateswill be required in the future.

This study found that the circulating vitrification bag methodwas able to maintain the morphological integrity of the vitrifiedand rewarmed cell sheet. The survival rates of component cells andcomponents of the extracellular matrix were also maintained aftervitrification and were on par with those of the non-vitrified sheets.The recovery of cartilage damage using the chondrocyte sheet wasshown to involve bioactive substances secreted from the cell sheet,including transforming growth factor beta 1, melanoma inhibitoryactivity, and prostaglandin E2 [5,8,9]. We have already confirmed in

Fig. 3. Comparative analysis of the characteristics of rabbit chondrocyte sheets before and after vitrification. Comparison of the morphological appearance (aec) of rabbit chon-drocyte sheets and distribution of proteoglycan (def) and type II collagen (gei) in cross-sections of the cell sheets. a: There was no visible crack in the cell sheet vitrified by thecirculating vitrification bag method. The extracellular matrix of the vitrified cell sheet contained abundant proteoglycan (d) and type II collagen (g) with a dense and even dis-tribution. These characteristics of the vitrified cell sheets were similar to those of the cell sheets vitrified by the envelope method (b, e, h) and non-vitrified controls (c, f, i). (Scalebar ¼ 50 mm).

Table 2Structural maintenance and cell viability after rewarming of rabbit chondrocyte sheets vitrified by the circulating vitrification bag method.

Vitrification method No. of cell sheets recovered without fracture/No. of cell sheets examined (%) Cell viabilitya

Circulating vitrification bag method 7/7 (100) 91.0 ± 2.9% (n ¼ 7)Envelope method 7/7 (100) 89.5 ± 1.4% (n ¼ 7)Non-vitrified control 7/7 (100) 90.0 ± 3.0% (n ¼ 7)

a Mean ± S. D.

Table 3Structural maintenance and cell viability of rabbit chondrocyte sheets preserved for long periods in the vitrification storage box.

Storage period No. of cell sheets recovered without fracture/No. of cell sheets examined (%) Cell viabilityb

Non-vitrified control e 6/6 (100) 84.3 ± 1.8% (n ¼ 6)Vitrified controla e 5/5 (100) 80.1 ± 2.5% (n ¼ 5)Vitrified and stored 1 month 3/3 (100) 79.2 ± 2.6% (n ¼ 3)

6 months 3/3 (100) 81.2 ± 1.0% (n ¼ 3)

a Vitrified control represents the cell sheet that was rewarmed immediately after vitrification.b Mean ± S. D.

Fig. 4. Morphological appearance of rabbit chondrocyte sheets after long-term vitrification preservation. The morphological appearance of rabbit chondrocyte sheets that werestored for long periods in the vitrification storage box was observed. a: Non-vitrified control cell sheet. b: The cell sheet vitrified with the envelope method and immediatelyrewarmed (vitrified control). c, d: Intact morphology of the cell sheets vitrified in the vitrification storage box and stored in the LN container for one month (c) and six months (d).

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221220

A. Hayashi et al. / Regenerative Therapy 14 (2020) 215e221 221

our previous study that these factors could be generated fromvitrified and rewarmed rabbit chondrocyte sheets [5]. In the future,we need to check whether the cell sheet vitrified using the circu-lating vitrification bag method has the same cartilage damage re-covery function.

In the present study, we did not test the applicability of thecirculating vitrification bag method to other cell sheet types thanrabbit chondrocytes. The vitrification protocol used in the circu-lating vitrification bag method has been proven to be effective forcryopreserving human myoblast cell sheet [10]. It is, therefore,likely that the circulating vitrification bag method is applicable tovarious cell sheets.

In the development of vitrificationmethods for animal embryos,including those of humans, the minimum volume cooling (MVC)concept [11] has beenwidely used. By considering that the utility ofthe MVC concept has been proven in embryo vitrification, wedeveloped the cell sheet vitrification method based on this concept[4]. In methods based on the MVC concept, the sample is cry-opreserved along with a minimum volume of vitrification solution.By minimizing the amount of vitrification solution used, the speedof the temperature decrease or increase of the sample is maxi-mized. During the temperature fall and rise, the solution quicklypasses through the temperature range at which ice crystals aremore likely to form. In this way, the risk of ice crystal formation inthe solution is reduced [12], and a high sample survival rate can bemaintained.

On the other hand, one drawback of methods based on the MVCconcept is that they aremore likely to result in devitrification. A cellsheet vitrified with a small amount of vitrification solution de-vitrifies evenwith a slight rise in temperature. Devitrification undernon-optimal conditions may result in cell damage associated withice crystal formation in solution. Such devitrification tends to occurwhen pulling the vitrified cell sheet out of the LN container ortransferring it within the laboratory or between facilities. Here, wedeveloped a vitrification storage box that is effective in the pre-vention of devitrification in the abovementioned situations.Moreover, the vitrification storage box can be used in the entireprocess, from vitrification of the cell sheet to storage and trans-portation. The characteristics of the cell sheet vitrification methodthat we previously developed [4] allow it to induce vitrification byexposing the sample to LN vapor (approximately �150 �C) ratherthan by immersing it in LN. The internal temperature of the vitri-fication storage box filled with LN absorbents is maintained stablyat approximately �150 �C, which produces the desired vitrificationconditions and preservation conditions of the vitrified cell sheet.

5. Conclusions

In conclusion, the results of this study indicated that the circu-lating vitrification bag method was potentially capable of realizingthe clinical application of vitrified chondrocyte sheets. The vitrifi-cation storage box was also shown to be useful for the long-term

preservation of vitrified cell sheets, making the clinical applica-tion of cryopreserved chondrocyte sheets even more feasible.

Declaration of Competing Interest

Asuka Hayashi, Miki Maehara, Ayuko Uchikura and HitomiMatsunari declare that they have no conflict of interest. KazuakiMatsumura and Suong-Hyu Hyon are cofounders of Bioverde Inc.Masato Sato receives research funding from CellSeed Inc. HiroshiNagashima is a founder and shareholder of PorMedTec Co., Ltd.

Acknowledgments

This work was supported by the Meiji University InternationalInstitute for Bio-Resource Research (MUIIBR), and the ResearchProject for Practical Applications of Regenerative Medicine (No.JP19bk0104063 to MS) from the Japan Agency for Medical Researchand Development. The authors thank Mr. Y. Tokuyama for histechnical assistance.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.reth.2020.04.006.

References

[1] Sato M, Yamato M, Mitani G, Takagaki T, Hamahashi K, Nakamura Y, et al.Combined surgery and chondrocyte cell-sheet transplantation improvesclinical and structural outcomes in knee osteoarthritis. npj Regen Med 2019;4:4.

[2] Toyoda E, Sato M, Takahashi T, Maehara M, Okada E, Wasai S, et al. Tran-scriptomic and proteomic analyses reveal the potential mode of action ofchondrocyte sheets in hyaline cartilage regeneration. Int J Mol Sci 2019;21(1).

[3] Maehara M, Sato M, Toyoda E, Takahashi T, Okada E, Kotoku T, et al. Char-acterization of polydactyly-derived chondrocyte sheets versus adult chon-drocyte sheets for articular cartilage repair. Inflamm Regen 2017;37:22.

[4] Maehara M, Sato M, Watanabe M, Matsunari H, Kokubo M, Kanai T, et al.Development of a novel vitrification method for chondrocyte sheets. BMCBiotechnol 2013;13:58.

[5] Tani Y, Sato M, Maehara M, Nagashima H, Yokoyama M, Yokoyama M, et al.The effects of using vitrified chondrocyte sheets on pain alleviation andarticular cartilage repair. J Tissue Eng Regen Med 2017;11:3437e44.

[6] Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at -196 de-grees C by vitrification. Nature 1985;313:573e5.

[7] Matsumura K, Hyon SH. Polyampholytes as low toxic efficient cryoprotectiveagents with antifreeze protein properties. Biomaterials 2009;30:4842e9.

[8] Kaneshiro N, Sato M, Ishihara M, Mitani G, Sakai H, Mochida J. Bioengineeredchondrocyte sheets may be potentially useful for the treatment of partialthickness defects of articular cartilage. Biochem Biophys Res Commun2006;349:723e31.

[9] Hamahashi K, Sato M, Yamato M, Kokubo M, Mitani G, Ito S, et al. Studies ofthe humoral factors produced by layered chondrocyte sheets. J Tissue EngRegen Med 2015;9:24e30.

[10] Ohkawara H, Miyagawa S, Fukushima S, Yajima S, Saito A, Nagashima H, et al.Development of a vitrification method for preserving human myoblast cellsheets for myocardial regeneration therapy. BMC Biotechnol 2018;18(1):56.

[11] Hamawaki A, Kuwayama M, Hamano S. Minimum volume cooling method forbovine blastocyst vitrification. Theriogenology 1999;51(1):165.

[12] Wowk B. Thermodynamic aspects of vitrification. Cryobiology 2010;60:11e22.